RNA molecules that adopt specific three-dimensional structures can participate in numerous biologically important processes, including many specifically related to diseases such as cancer. Some RNAs are also capable of catalyzing chemical reactions, a role typically played by protein enzymes. Investigating RNA structure, folding, and catalysis is a major effort of modern biochemistry. For these studies, it is desirable to incorporate modified nucleosides at particular positions of RNA. Modified nucleosides are useful both to allow a detailed atomic-level understanding of RNA structure-function relationships and to enable incorporation of biophysical probes such as fluorescent labels. However, current techniques do not allow dependable synthesis of large, site-specifically modified RNAs. In this proposal, a comprehensive approach will be developed for ligation of smaller RNA fragments, which may themselves incorporate modifications. The ligation reaction will join two RNAs that have readily obtained functional groups at the ligation junction (e.g., 2',3'-cyclic phosphate and 5'-hydroxyl). The reaction will be catalyzed by divalent metal-dependent deoxyribozymes (DNA enzymes). These will be identified by a new in vitro selection .strategy, starting either from random DNA pools or from DNA pools biased towards sequences of RNA-cleaving DNA enzymes. The selected deoxyribozymes are anticipated to be efficient, general, and reliable tools for sequence-specific ligation of RNA. The new deoxyribozymes will be fully characterized biochemically and structurally. This will allow their optimal use for practical RNA ligations, and it will expand our knowledge of how nucleic acids can accelerate chemical reactions. The results of this research will have implications for designing therapeutic nucleic acid enzymes directed towards specific biological targets in vivo. The newly identified deoxyribozymes will be applied to perform previously unachievable experiments in RNA structure, folding, and catalysis that require incorporation of modified nucleosides.